Molecular cloning and characterization of CYP80G2, a cytochrome p450 that catalyzes an intramolecular C-C phenol coupling of (S)-reticuline in magnoflorine biosynthesis, from cultured Coptis japonica cells
Molecular cloning and characterization of CYP80G2, a cytochrome p450 that catalyzes an intramolecular C-C phenol coupling of (S)-reticuline in magnoflorine biosynthesis, from cultured Coptis japonica cells
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DOI：
10.1074/jbc.m705082200
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发表时间：
2008-04-04
影响因子：
4.8
通讯作者：
Sato, Fumihiko
中科院分区：
文献类型：
--
作者：
Ikezawa, Nobuhiro;Iwasa, Kinuko;Sato, Fumihiko
细胞色素P450（P450）在植物次生代谢的氧化反应中起着关键作用。它们中的一些催化除了标准羟基化以外的独特反应，增加了植物次生代谢物的结构多样性。在异喹啉生物碱的生物合成中，有几种独特的P450反应被报道，如亚甲二氧基桥的形成、分子内C-C酚偶联和分子间C-O酚偶联反应。本文报道了从培养的黄连细胞表达序列标签文库中分离并鉴定了一个C-C酚偶联P450 cDNA（CYP 80 G2）。结构分析表明，CYP 80 G2与参与小檗碱生物合成的分子间C-O酚偶联P450--匍匐小檗CYP 80 A1具有高度的氨基酸序列相似性。在酵母中的异源表达表明，CYP 80 G2具有分子内C-C酚偶联活性，可从（S）-牛心果碱（苄基异喹啉型）产生（S）-corytuberine（阿朴啡型）。尽管这种有趣的反应，重组CYP 80 G2显示出典型的P450特性：其C-C酚偶联反应需要NADPH和氧，并被典型的P450抑制剂抑制。基于对底物特异性的分析，对这种独特的反应机理和底物识别进行了讨论。CYP 80 G2可能参与木兰花碱的生物合成。japonica，基于重组C. japonica S-腺苷-L-甲硫氨酸：乌药碱N-甲基转移酶可将（S）-紫堇叶碱转化为木兰花碱。
Cytochrome P450s (P450) play a key role in oxidative reactions in plant secondary metabolism. Some of them, which catalyze unique reactions other than the standard hydroxylation, increase the structural diversity of plant secondary metabolites. In isoquinoline alkaloid biosyntheses, several unique P450 reactions have been reported, such as methylenedioxy bridge formation, intramolecular C-C phenol-coupling and intermolecular C-O phenol-coupling reactions. We report here the isolation and characterization of a C-C phenol-coupling P450 cDNA (CYP80G2) from an expressed sequence tag library of cultured Coptis japonica cells. Structural analysis showed that CYP80G2 had high amino acid sequence similarity to Berberis stolonifera CYP80A1, an intermolecular C-O phenol-coupling P450 involved in berbamunine biosynthesis. Heterologous expression in yeast indicated that CYP80G2 had intramolecular C-C phenol-coupling activity to produce ( S)-corytuberine ( aporphinetype) from (S)-reticuline (benzylisoquinoline type). Despite this intriguing reaction, recombinant CYP80G2 showed typical P450 properties: its C-C phenol-coupling reaction required NADPH and oxygen and was inhibited by a typical P450 inhibitor. Based on a detailed substrate-specificity analysis, this unique reaction mechanism and substrate recognition were discussed. CYP80G2 may be involved in magnoflorine biosynthesis in C. japonica, based on the fact that recombinant C. japonica S-adenosyl-L-methionine: coclaurine N-methyltransferase could convert (S)-corytuberine to magnoflorine.